Since the early 1990s, expansion of unstable nucleotide (microsatellite) repeats, notably trinucleotide repeat was identified as a novel mutational mechanism underlying certain human diseases. Over the years, several additional developmental and neuromuscular disorders were identified to be caused by either an insertion or a duplication of trinucleotide repeats as well as unstable tetra-, penta-, hexanucleotide, and longer repeats (Mirkin 2007). This insertion or duplication of polynucleotide repeats can induce a protein loss of function, a RNA toxic gain of function or a protein toxic gain of function leading to the disorder. Examples of such disorders include Huntington disease, inherited ataxias, fragile X syndrome, myotonic dystrophy a common genetic muscular dystrophy, a group of dominantly inherited ataxias, and most recently an unstable hexanucleotide repeat in the C9ORF72 gene as a frequent cause of frontotemporal dementia/amyotrophic lateral sclerosis (DeJesus-Hernandez, Mackenzie et al. 2011; Renton, Majounie et al. 2011) (see for review (Nelson, Orr et al. 2013)).
Treatment options for most of repeat expansion disorders are very limited. One of the most attractive therapeutic strategies envisaged for various neurodegenerative diseases is gene therapy. Indeed, several strategies to turn off expression of repeat expanded have been developed. In particular, silencing the mutant gene using RNA interference technology within cell has been realized for preventing the toxic function of the protein or RNA (Wang, Liu et al. 2005; Machida, Okada et al. 2006; DiFiglia, Sena-Esteves et al. 2007). However, basically the design of RNA interference does not allow the distinction between the normal and repeat expansion sequences and induce simultaneous reduction of both the mutant and wild type gene (Caplen, Taylor et al. 2002). However, the huntingtin protein is widely expressed and is required for neuronal function and survival in the brain (Duyao, Auerbach et al. 1995; Dragatsis, Levine et al. 2000). Thus, it is important to reduce specifically expression of the mutant gene, while leaving the expression of the wild type protein unaffected.
Recently, Zinc Finger proteins were designed to bind poly-trinucleotides repeat of the huntingtin gene, responsible for the Huntington disease. Zinc fingers were concatenated into long chains with appropriate linker to obtain an optimal configuration for repressing preferably the repeat expanded huntingtin gene compared with the shorter repeats. This strategy allows more efficient repression of mutant gene expression compared to wild type gene. However it has not been known whether the repression would be sufficient to reduce protein levels for gene therapy (Garriga-Canut, Agustin-Pavon et al., International application: WO2013/130824).
A previous study (Richard, Dujon et al. 1999) has suggested that Induction of a cleavage event within the repeat sequence was associated with contraction of trinucleotide repeat arrays, which may be explained by two different mechanisms: (1) the two ends of the break are available to invade the template, but they can invade at any location within the template, since they carry repeated sequences that are homologous to the template; or (2) only one end invades the template and the newly synthesized strand is displaced from its template, but can anneal with the other end containing repeats (Richard, Dujon et al. 1999). However, due to the highly frequency of repeat sequences within the genome, engineered DNA binding nuclease designed to be specific to said repeat sequences, are likely to induce off-site mutagenesis at several positions throughout the human genome. Consequently, the ability to create a cleavage in the repeat sequence only at the desired genomic position would be highly desirable.
To overcome the above limitations, the present inventors have developed a genetic therapeutic strategy to decrease the number of expanded polynucleotide repeats by using DNA binding nucleases, while maintaining the integrity of the genome and functionality of the corrected gene. This strategy mainly relies on the design of the DNA binding nucleases along with the selection of genome sequences to specifically target the repeat sequence associated with the triplet repeat disorders.